Keith Nykamp et al. (2006) Development & Evolution Meeting "The Conserved La Motif Protein, LARP-1, Negatively Regulates MAPK Signalling in the Germ Line"

Judith Kimble, Keith Nykamp

[Development & Evolution Meeting, 2006]

The human La protein was first described as an autoantigen present in the sera of systemic lupus erythematosus (SLE) and Sjogren's patients. Since then, a large family of La-related proteins has been described (Wolin & Cedervall, 2002). La family members are all characterized by a highly conserved La Motif (LaM), which has RNA-binding activity. Typically, LaM family members, including human La and yeast Lhp1p, harbor additional RNA Recognition Motifs (RRMs) and localize predominately to the nucleus. Both La and Lhp1p bind RNA polymerase III transcripts (i.e. tRNAs, 5S rRNAs and snRNAs) and direct their maturation. In contrast, two atypical yeast LaM proteins, Sro9p and Slf1p, do not have any other recognizable RNA-binding domains and are predominately cytoplasmic. Sro9p and Slf1p are believed to play a role in mRNA metabolism, although it is unclear what their RNA targets and exact mechanism of control might be (Sobel and Wolin, 1999). The Caenorhabditis elegans genome encodes three LaM family members: C44E4.4, T12F5.5 and R144.7/larp-1. Of the three, LARP-1 is most closely related to yeast Sro9p and Slf1p. Initial RNAi experiments indicated that LARP-1 might be important for oogenesis. To confirm the RNAi results, we isolated larp-1(q783), a deletion mutant that eliminates expression of LARP-1 protein. We find that larp-1(q783) hermaphrodites are self-fertile, but have more developing oocytes in the proximal gonad than normal. We have also found active MAP kinase to be more abundant in larp-1(q783) germ lines than in wild-type. Our working model is that LARP-1 negatively regulates MAPK activity to ensure proper oogenesis.

Esther Zanin et al. (2005) International Worm Meeting "Studying the function of the PAR-3 complex in the early C. elegans embryo"

Monica Gotta, Esther Zanin

[International Worm Meeting, 2005]

Cell polarity is a crucial biological process that is essential to generate cell diversity. The anterior-posterior axis of the C. elegans embryo is determined by the entry of the sperm. The earliest polarity markers known are the conserved partitioning defective (PAR) proteins that localize asymmetrically on the cell cortex in the one cell embryo after fertilization. A protein complex consisting of PAR-3, PAR-6 and aPKC-3 (atypical protein kinase C 3) is enriched on the anterior cortex. Together PAR-3, PAR-6 and aPKC-3 (PAR-3 complex) determine the orientation of the first mitotic spindle along the anterior-posterior axis and the displacement of the spindle during anaphase toward the posterior of the embryo. In addition the PAR-3 complex has been suggested to regulate the cell lineage specific expression of cell fate determinants. Although there is clear support for the requirement of the PAR-3 complex in establishing and maintaining anterior-posterior polarity in the early C. elegans embryo, much less is known about its molecular functions. Therefore we want to identify phosphorylation targets of PKC-3 in C. elegans and investigate how their activity is modulated by PKC-3 in order to generate cell diversity. We performed a yeast two-hybrid screen using a C. elegans embryonic cDNA library and the kinase-dead kinase domain of PKC-3 as a bait. We identified a LA-domain containing protein as an interactor of PKC-3. The LA-domain is a putative RNA-binding domain but the molecular function of this protein is unknown. Interestingly SRO9, a S. cerevisiae LA-domain containing protein, shows a genetic interaction with tropomyosin 1 and RHO3. This observation, together with other data in S. cerevisiae, indicates that SRO9 is required for the stabilization of actin filaments. Depletion of LA by RNAi results in less than 10% embryonic lethality. However, the embryos show a strong posterior meeting of the paternal and maternal pronuclei, a reduced AB size and they are small. Interestingly we found that reducing the level of the LA by RNAi can partially rescue par-2 (it5ts) lethality. This suggests that the LA-domain protein could function either as a regulator or an effector of the PAR-3 complex. In order to understand if the reduction of the LA-domain protein influences the localization and/or levels of the PAR-3 complex we will analyze PAR-3 and PAR-6 immuno-staining in par-2 (it5ts) mutant embryos where the LA has been depleted by RNAi. Finally we aim to reveal if the LA-domain protein regulates F-actin stability via tropomyosin by testing its genetic interactions further. Current progress on the project will be presented at the meeting.

Keith Nykamp et al. (2008) Development & Evolution Meeting "LARP-1 Localizes to Germline P Bodies and Attenuates RAS-MAPK signaling During Oogenesis"

Myon-Hee Lee, Judith Kimble, Keith Nykamp

[Development & Evolution Meeting, 2008]

RNA regulators are critical for animal development, especially in the germ line. In this work, we focus on C. elegans LARP-1, a representative of one La-related protein (Larp) cluster that is broadly conserved among eukaryotes. LARP-1 possesses a signature La motif, which is an ancient RNA-binding domain, plus another conserved motif we have dubbed the LARP1 domain. LARP-1 binds RNA in vitro via the La motif and the LARP1 domain. A larp-1 null mutant has germline defects reminiscent of hyperactive Ras-MAPK signaling; genetic interactions indicate that LARP-1 normally attenuates Ras-MAPK signaling in the germ line; and mRNAs encoding certain Ras-MAPK signaling components are elevated in larp-1(0) mutant germ lines. LARP-1 co-localizes with cytoplasmic granules called P bodies, which have been implicated in mRNA degradation. We propose that LARP-1 affects the stability of selected mRNAs via its association with germline P bodies.

Keith Nykamp et al. (2005) International Worm Meeting "The conserved RNA-binding protein, LARP-1, interacts with the atypical poly(A) polymerase GLD-2 and regulates meiotic progression"

Keith Nykamp, Liaoteng Wang, Judith Kimble

[International Worm Meeting, 2005]

The adult hermaphrodite germ line is spatially organized with proliferative stem cells localized to the distal end and fully differentiated sperm and oocytes at the proximal end. In between, meiotic nuclei progress from pre-meiotic S phase and early meiotic prophase through pachytene and diplotene/diakinesis. This developmental pattern is established, in a large part, by a combination of Notch signaling, RNA regulators and MAP kinase activity. Among RNA regulators, GLD-2 and GLD-3 work together to promote entry into the meiotic cell cycle. GLD-2 is an atypical poly(A) polymerase that lacks any apparent RNA-binding motif, and GLD-3 is a KH RNA-binding protein and GLD-2 binding partner. We have focused on a different GLD-2 binding partner to explore the idea that GLD-2 is recruited to distinct mRNAs by interacting with distinct RNA-binding proteins. In a yeast two-hybrid screen for GLD-2 interacting proteins, 42% (50/118) of the positive isolates corresponded to the larp-1 (La-related protein) gene. larp-1 encodes a large protein with a single La motif. The La motif has RNA-binding activity in vitro with a preference for 3 termini, is found in multiple proteins in eukaryotes and has been associated with a wide variety of functions. We will provide genetic, biochemical and cytological data to support the idea that GLD-2 and LARP-1 function together to control the transition from pachytene to diplotene/diakinesis of developing oocytes.

Yarbrough PO et al. (1985) International C. elegans Meeting "isolation of a GAPDH gene from the nematode C elegans."

Klass MR, Hayden MA, Dunn LA, Hecht RM, Yarbrough PO, Vermersch PS

[International C. elegans Meeting, 1985]

Sommer, Ralf J. (2011) International Worm Meeting "Molecular phylogeny and divergence time estimates for beetle-associated Pristionchus nematodes."

Sommer, Ralf J.

[International Worm Meeting, 2011]

Pristionchus pacificus has been established as a model system in evolutionary developmental biology (evo-devo) and for comparison to C. elegans. Recent studies expanded the evo-devo work to ecology and population genetics. P. pacificus and related species have a well-defined association with scarab beetles: Sampling of more than 20,000 scarab beetles from around the world resulted in the isolation of more than 8,000 Pristionchus isogenic female lines. Mating experiments and molecular sequence analysis identified 26 species to date. We provide a molecular phylogenetic framework based on nearly 11,000 characters. Studies on La Reunion, an Island with a unique P. pacificus biodiversity hotspot, provide a case study to link population genetics with ecology and evo-devo. We obtained more than 400 wild isolates of P. pacificus and carried out microsatellite analysis of 21 markers. These studies indicate four major biogeographic clades of P. pacificus. La Reunion represents the only geographic area with P. pacificus strains being present in all four clades. This finding is best explained by P. pacificus invading the island independently with different beetle vectors, such as Oryctes, Amneidus and Maladera (Herrmann et al., 2010). We assessed the molecular phylogeny of P. pacificus and used mutation accumulation line approaches to provide divergence time estimates. Mutation rates were assessed first, by mitochondrial DNA analysis and second, from representative microsatellite markers. Mitochondrial DNA analysis suggests a minimal divergence time for P. pacificus of 105 to 106 generations (Molnar et al., 2011). Similarly, microsatellite markers are used to robustly provide minimal divergence time estimates for closely related strains from La Reunion. This includes a first attempt for a serious analysis of the population size of P. pacificus.

Shivaiah, Shashikumar et al. (2013) International Worm Meeting "PUFA therapy ameliorates Parkinson disease like symptoms."

Shivaiah, Shashikumar, Golgodu Krishnamurty, Rajanikant

[International Worm Meeting, 2013]

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting about 1 - 2% of the population over the age of 65. PD encompasses a spectrum of core clinical features such as rigidity, bradykinesia, tremor at rest and postural instability. However, PD is a heterogeneous disorder, as many patients also develop cognitive dysfunctions, including anxiety, depression and dementia or abnormalities in olfactory and visual perception. Pathologically, PD is characterized by the specific and massive loss of dopamine (DA) containing neurons in the Substantia Nigra pars compacta and aggregation of protein alpha-Synuclein levels. Poly unsaturated fatty acid (PUFA) are highly abundant in the brain and omega-3 and omega-6 PUFA are essential structural components of the cell membrane phospholipids. Omega-3 fatty acids are dietary essentials, and the current low intakes in most modern developed countries are believed to contribute to a wide variety of physical and mental health problems. In the present study, the chemically induced PD symptoms in transgenic Caenorhabditis elegans UA44 (baln11;Pdat-1::Pdat-1::GFP) exposed to various concentration of 6-Hydroxydopamine (6-OHDA) by exposing from the egg stage to adult larva with and with out supplementation of PUFA Linoleic acid (LA) along with food source. The results were confirmed by various parameters, the decreased locomotion being normalized to like control by counting number of body bends (29 and 42% decrease in locomotion in 5 and 25mM 6-OHDA and 20% protection by LA against 6-OHDA), body thrashing and cognition enhancement by PUFA (LA) supplemented groups are well evidenced. The main markers of PD symptoms alpha Synuclein accumulation and degeneration of dopamine neuron were similarly reduced, as evidenced by fluorescence intensity and microscopic analysis. The mitochondrial enzymes decreased were measured such as Succinate dehydrogenase, complex I and IV, total thiols and cytochrome c activity. In conclusion, the nematode model C. elegans with its simple nervous system and GFP tagged dopaminergic neuron offer an valuable tool for studying the pathogenesis of Parkinson disease and evaluate the anti-Parkinsonian effects of PUFA molecules.

Marisa L Foehr et al. (2003) International Worm Meeting "The C. elegans Schnurri homolog SMA-9 mediates cell type specific LIN-12/Notch signaling in the postembryonic mesoderm."

Yuan Jiang, Marisa L Foehr, Maegan Rivard, Rachel Fairbank, Jun Liu

[International Worm Meeting, 2003]

Many developmental processes including the specification of cell fate are mediated by highly conserved cell-cell signaling pathways. While the general mechanisms by which these signaling pathways function are well defined, the mechanisms mediating how the same signaling pathway can regulate different processes in different cellular context are less well understood. We are interested in a molecule, SMA-9, that functions specifically with the Notch signaling pathway to regulate cell fate specification in the C. elegans postembryonic mesodermal lineage, the M lineage. In the M lineage, sma-9 mutants specifically affect the decision between two cell fates, the sex myoblasts and the coelomocytes. In addition, sma-9 mutant animals are smaller than wild type. Work from the lab of Cathy Savage-Dunn has shown that sma-9 encodes the C. elegans homolog of the Drosophila protein Schnurri, a known regulator of the TGFb signaling pathway. The Savage-Dunn lab has also shown that SMA-9 does interact with the TGFb signaling pathway in regulating body size. However, the function of SMA-9 in M lineage fate specification appears to be independent of the TGFb signaling pathway. We have shown that sma-9 genetically interacted with LIN-12/Notch specifically in the M lineage. In the yeast two-hybrid system, SMA-9 interacted with LAG-1, a downstream transcription factor of the Notch signaling pathway. These suggest that SMA-9 might mediate different signaling pathways in a cell-type specific manner. Consistent with this, SMA-9 has different splicing isoforms. Therefore, we hypothesize that the different SMA-9 isoforms might mediate different signaling pathways in different cell types. To test the above hypothesis, we have generated isoform specific antibodies to two of the three SMA-9 isoforms and examined the expression pattern of SMA-9 using these antibodies. Both SMA-9 isoforms are present in the nucleus. However, they showed different patterns of expression. We are further characterizing the function of each isoform in the M lineage by examining their expression in the M lineage and by testing their interaction with LAG-1 in solution and in worm extracts. A suppressor screen for sma-9 has also been carried out in an attempt to isolate other factors that interact with SMA-9.

Gimond, Clotilde et al. (2011) International Worm Meeting "Phenotypic characterization of the tropical hermaphroditic species Caenorhabditis sp. 11."

Braendle, Christian, Mauri, Alessandra, Ferrari, Celine, Grimbert, Stephanie, Vigne, Paul, Gimond, Clotilde, Callemeyn-Torre, Nicolas, Poullet, Nausicaa

[International Worm Meeting, 2011]

The majority of Caenorhabditis species exhibits a male-female mode of reproduction, and hermaphroditism has evolved only twice, in C. elegans and C. briggsae. Recently, a third hermaphroditic species, C. sp. 11, has been identified by Marie-Anne Felix (strain JU1373, La Ruunion). Since then, the isolation of more than 30 additional C. sp. 11 wild isolates from La Reunion, Cap Verde, Puerto Rico, Hawaii, Guadeloupe and mainland South America (French Guiana, Brazil) indicates that this species occurs primarily, if not exclusively, in tropical regions. Here we present the results of our current phenotypic characterization of recently collected wild isolates of C. sp. 11 with the aims (i) to quantify the degree of intraspecific variation and its correlation with geographic origin, (ii) to test for plasticity and genotype-by-environment interactions in reproductive traits at different temperatures and (iii) to ask how C. sp. 11 phenotypic characteristics differ from the ones observed in C. elegans and C. briggsae. This survey focuses mainly on germline and reproductive features (e. g. germ cell number, sperm number and size, offspring number) and basic life history traits (e.g. male production, propensity to enter dauer, longevity). Our initial observations confirm that C. sp. 11 isolates are overall much more heat-tolerant (and less cold-resistant) than C. elegans isolates, e.g. they maintain their reproductive output at 27 deg C similar to tropical C. briggsae strains. Interestingly, there is also substantial variation in reproductive output among different C. sp. 11 isolates, and many isolates have a much reduced offspring number relative to N2 and AF16. This reduction in offspring number correlates with a much reduced sperm and germ cell number, and we are currently testing to what extent germline size and reproductive output are modulated by temperature.

Wallenfang MR et al. (1997) International C. elegans Meeting "ISOLATION OF MUTANTS DEFECTIVE IN TRANSCRIPTIONAL REPRESSION IN THE EMBRYONIC GERM LINEAGE OF C. ELEGANS"

Seydoux GC, Wallenfang MR, Dunn MA

[International C. elegans Meeting, 1997]

Previous work has suggested that transcription of embryonic mRNAs is repressed in the early germ lineage1. One factor which has been shown to be involved in this repression is the PIE-1 protein; in pie-1(-) mutants, transcription of embryonic mRNAs is activated in germline blastomeres. We have begun a genetic screen to identify other factors which may be involved in this process. This screen makes use of a pes-10::gfp transgene that expresses GFP in all somatic lineages, but not in the germ lineage, of early embryos. Mutations that activate transcription in germline blastomeres are readily recognized as mutations that cause GFP to be expressed in all cells. From 5500 F1s screened, we have isolated three maternal effect lethal mutants which express GFP in all embryonic blastomeres, and two in which no embryonic GFP expression is seen. Preliminary characterization suggests that some of these mutants represent alleles of previously identified par genes. Further characterization of these mutants is underway, including phenotypic analysis, and immunostaining for PIE-1, P granules, and phosphorylated isoforms of RNA polymerase II. 1. Seydoux et al. (1996). Nature 382, 713-716; abstract by Dunn and Seydoux, IWM '97.